Bottom Line:
In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity.While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis.Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

Background: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers.

Methodology/principal findings: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis.

Conclusions/significance: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

pone-0001815-g006: (A) Influence of glutamine on the conformation of the PfPdx1 DKK mutant.Static light scattering analysis of the plasmodial Pdx1 DKK protein was performed in the presence of 10 mM glutamine. The proposed structural conformations are given above the peak fractions. (B) Affect of mutations on Pdx1 interactions between hexamers. Monomers coloured in green and violet belong to the upper hexameric crown and the monomer coloured in salmon belongs to the lower hexameric crown. Mutations in R85, H88 and E91 of the upper crown affect the conformation of loop 113–119, which interacts with loop-helix 179–191 in the lower crown. Helix 88–98 interacts directly with Pdx2 (not shown).

Mentions:
Interestingly, addition of 10 mM glutamine (Q) to the buffer used in size exclusion chromatography, resulted in an approximate 50% shift in the assembly of the hexameric formation of the DKK mutant towards the dodecameric structure as estimated from the molecular mass plot obtained by SLS (Fig. 6A). A similar effect was obtained by the addition of 10 mM glutamic acid (E) as well as asparagine (N), although to a lesser extent. In contrast alanine (A) does not affect dodecamer formation.

pone-0001815-g006: (A) Influence of glutamine on the conformation of the PfPdx1 DKK mutant.Static light scattering analysis of the plasmodial Pdx1 DKK protein was performed in the presence of 10 mM glutamine. The proposed structural conformations are given above the peak fractions. (B) Affect of mutations on Pdx1 interactions between hexamers. Monomers coloured in green and violet belong to the upper hexameric crown and the monomer coloured in salmon belongs to the lower hexameric crown. Mutations in R85, H88 and E91 of the upper crown affect the conformation of loop 113–119, which interacts with loop-helix 179–191 in the lower crown. Helix 88–98 interacts directly with Pdx2 (not shown).

Mentions:
Interestingly, addition of 10 mM glutamine (Q) to the buffer used in size exclusion chromatography, resulted in an approximate 50% shift in the assembly of the hexameric formation of the DKK mutant towards the dodecameric structure as estimated from the molecular mass plot obtained by SLS (Fig. 6A). A similar effect was obtained by the addition of 10 mM glutamic acid (E) as well as asparagine (N), although to a lesser extent. In contrast alanine (A) does not affect dodecamer formation.

Bottom Line:
In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity.While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis.Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.

Background: Plants, fungi, bacteria and the apicomplexan parasite Plasmodium falciparum are able to synthesize vitamin B6 de novo, whereas mammals depend upon the uptake of this essential nutrient from their diet. The active form of vitamin B6 is pyridoxal 5-phosphate (PLP). For its synthesis two enzymes, Pdx1 and Pdx2, act together, forming a multimeric complex consisting of 12 Pdx1 and 12 Pdx2 protomers.

Methodology/principal findings: Here we report amino acid residues responsible for stabilization of the structural and enzymatic integrity of the plasmodial PLP synthase, identified by using distinct mutational analysis and biochemical approaches. Residues R85, H88 and E91 (RHE) are located at the Pdx1:Pdx1 interface and play an important role in Pdx1 complex assembly. Mutation of these residues to alanine impedes both Pdx1 activity and Pdx2 binding. Furthermore, changing D26, K83 and K151 (DKK), amino acids from the active site of Pdx1, to alanine obstructs not only enzyme activity but also formation of the complex. In contrast to the monomeric appearance of the RHE mutant, alteration of the DKK residues results in a hexameric assembly, and does not affect Pdx2 binding or its activity. While the modelled position of K151 is distal to the Pdx1:Pdx1 interface, it affects the assembly of hexameric Pdx1 into a functional dodecamer, which is crucial for PLP synthesis.

Conclusions/significance: Taken together, our data suggest that the assembly of a functional Pdx1:Pdx2 complex follows a defined pathway and that inhibition of this assembly results in an inactive holoenzyme.